Regulatory element for heterologous protein production in the fruiting body of filamentous fungi

ABSTRACT

The present invention provides compositions and methods for regulating expression of nucleotide sequences in fungi. Compositions are novel nucleotide sequences for a tissue preferred promoter isolated from the  Agaricus bisporus  lectin gene. The sequences drive expression preferentially to fruit body tissue. A method for expressing a nucleotide sequence in fungi using the regulatory sequences disclosed herein is provided. The method comprises transforming a fungal cell to comprise a nucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed fungus from the transformed cell.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, moreparticularly to regulation of gene expression in fungi. The novelcompositions and methods can be used to improve fungal species byrecombinant technologies known to those of skill in the art, such as theimproved pathogen, pest, and pesticide resistance, yield quantity andquality, extended produce shelf life, improved culinary, nutritional,and medicinal value, and the like, as well as the commercial productionof heterologous proteins.

BACKGROUND OF THE INVENTION

About 40% of the commercially available enzymes are derived fromfilamentous fungi. Lowe, Handbook of Applied Mycology. FungalBiotechnology (eds.) Arora, D. K. Elander, R. P. & Mukerji, K. G.681-708 (Marcel Dekker, New York; 1992). These enzymes are usuallyproduced by species of the genera Aspergillus and Trichoderma. Becausethey secrete large amounts of protein into the medium, they can be grownin large-scale fermentation, and they are generally accepted as safe forthe food industry.

General problems associated with the commercial cultivation of mushrooms(Agaricus bisporus) include diseases caused by pathogens likeVerticillium fungicola (dry bubble), Trichoderma aggressivum (greenmold), Pseudomonas tolaasii (blotch), and dsRNA viruses (La Francedisease and patch disease), the major insect pest [sciarid fly(Lycoriella mali)], an extremely short shelf life of the product relatedto bacterial spoilage and rapid senescence, and browning (bruising) ofthe fruit body associated with the action of endogenouspoly-phenoloxidases (PPO, like tyrosinase). To further improve productquality, conventional breeding programs for A. bisporus have been onlymoderately successful and may not be sufficient in the long term. Thisis because conventional breeding techniques for fungi are highly timeconsuming, and because the genetic variation in commercially availablestrains is limited, offering little advancement by selection (Horgen etal. “Homology between mitochondrial DNA of A. bisporus and an internalportion of a linear mitochondrial plasmid of Agaricus bitorquis” CurrGenet. 1991. 19:495-502).

In the case of A. bisporus, the main obstacle to effective breedingstrategies is the rather abnormal life cycle involving the unusualsimultaneous segregation of either parental nucleus into onebasidiospore. After outgrowth of this basidiospore, heterokaryoticmycelium is formed containing nuclei and genetic characteristics that donot differ from those present in the parental mycelium. In addition,only little recombinational activity is observed during meiosis(Summerbell et al. Genetics, October 123(2) 1989 pp. 293-300).

To overcome the limitations of conventional breeding, investigators allover the world have attempted to develop and improve transformationmethods for commercial mushrooms, such as A. bisporus, for theintroduction of novel characteristics. For other fungi, as well asplants, animals, and bacteria, the application of gene transfertechnology is quite common and has already resulted in commercialapplication.

In order to enhance the economies of protein production inmicroorganisms, such as fungi, there have been substantial efforts toimprove the efficiency of transcription and translation, maximize theproportion of total protein directed to production of the desiredproduct, enhance the viability of the modified host, and improve theefficiency with which the modified host may be obtained. The primarypromoter used in fungal transformation to date is theglyceraldehyde-3-phosphate dehydrogenase (gpd) promoter. Using strongpromoters to express heterologous genes in appropriate hosts is a majorstrategy in biotechnological applications. The gpd promoter is a strongpromoter that can be induced by any carbon source and has been widelyused in the expression of heterologous proteins in Saccharomycescerevisiae, Pichia pastoris and other yeasts.

The gpd genes have also been cloned from basidiomycetous fungi,including Schizophyllum commune, Phanerochaete chrysosporium, Agaricusbisporus (Harmsen et al., 1992), and Lentinula edodes (Hirano et al.,1999). Among these mushrooms, genetic transformation using homologousgpd promoter was reported to be successful only in A. bisporus,Flammulina velutipes and L. edodes (Hirano et al., 2000, Kuo et al.,2004, van de Rhee et al., 1996). Although heterologous promoters havebeen used for the expression of drug-resistant marker genes, the genetictransformation is not sufficient to express heterologous genes(Ruiz-Diez, 2002). To sufficiently and effectively express aheterologous gene, it is important for a host cell to recognize thepromoter sequence by its transcriptional machinery. Chun-Yi Kuo et al.demonstrated that a heterologous gene, hygromycin B phosphotransferasegene (hpt), can be expressed in F. velutipes (Kuo et al., 2004).However, it was found that although the gpd genes in basidiomycetousfungi are highly similar, these gene differ significantly in theirpromoter regions.

As illustrated by the foregoing, there is a continuing need in the artfor development of effective, convenient, and expeditious fungaltransformation systems and gene expression components.

It is thus an object of the present invention to provide atransformation system and in particular a strong regulatory element forfungi that will accomplish the foregoing need.

A further object of this invention is to provide mechanisms forapplication of transgenic techniques such as those applied to bacteria,non-filamentous fungi (yeast), plants, and animals to increase yield,disease, and pest resistance, product quality, shelf life, or culinary,nutritional, or medicinal value, to produce heterologous proteinscommercially, or other such protocols.

It is yet another object of the invention to provide regulatory elementscapable of driving high level protein accumulation of operably linkedsequences in the fruit body of fungi, as well as tissues in plant, oranimal cells.

It is yet another object of the invention to provide regulatory elementspolynucleotide constructs, vectors, and transformed cells for use insuch transgenic protocols.

Other objects of the invention will become apparent from the descriptionof the invention that follows.

SUMMARY OF THE INVENTION

The invention comprises an isolated regulatory element/promotercomprising a polynucleotide sequence selected from the group consistingof a) a polynucleotide sequence comprising the nucleic acid sequence asshown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; and b) afragment of the polynucleotide sequence of a) capable of regulatingtranscription of an operably linked polynucleotide molecule in a fungalcell.

The invention also comprises a polynucleotide construct comprising thepromoter of the invention operably linked to a heterologouspolynucleotide molecule.

The invention also includes a transformed cell comprising the constructof the invention as well as methods of transformation using theconstructs of the invention and heterologous protein products producedthereby.

The invention is directed to a promoter from an A. bisporuslectin-encoding gene, useful as a regulatory region and providing forexpression of a nucleotide sequence of interest. In an embodiment, theaccumulation of a heterologus protein or nucleotide sequence of interestis driven to the fruit body tissue. The invention is further directed tofunctional fragments that drive expression of a heterologus protein ornucleotide sequence of interest—in the fruit body tissue. Expressioncassettes that incorporate the promoter driving expression of anucleotide sequence, plants, fungi or bacterial cells expressing same,and methods of use in modulating expression of nucleotides sequences arewithin the scope of the invention.

DESCRIPTION OF FIGURES

FIG. 1 is a 2-D gel analysis of proteins extracted from Agaricusbisporus fruiting bodies. Protein separation on 2-D SDS PAGE wasperformed according to the manufacturer's recommendation using IPGdrystrips for the first dimension isoelectric focusing, and NuPAGEpre-cast gel system from Invitrogen for the 2nd dimension separation.The numbers identify protein spots excised for MS and MS/MS analysis.

FIG. 2 is a map of binary vector pAGN-750 which includes the promoter ofthe invention linked to a reporter gene.

FIG. 3 is a photograph showing expression of a GUS reporter gene in thevegetative mycelium of pAGN-750 events grown on filter paper.

FIG. 4 is a photograph showing GUS staining of fruiting body slices forindependent transgenic events of pAGN-750. Sample 1=750-22B; sample2=750-16A; sample 3=750-6B; sample 4=750-11C; sample 5=750-7A; sample6=750-13A; sample 7=750-8A; sample 8=750-23A

FIG. 5 is a map of Binary vector pAGN-755 which includes the promoter ofthe invention linked to a target gene of interest.

FIG. 6 depicts the sequences of the lectin promoter. 5′ end DNAsequences in each vector are underlined and the corresponding vectoridentified. All vectors share the same 3′ sequence as presented.pAGN-755 and pAGN-750 share the identical sequence.

FIG. 7 shows the GUS staining of liquid mycelium culture of the eventsgenerated from truncated promoters shown in Table 5.

DETAILED DESCRIPTION OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

In accordance with the invention, nucleotide sequences are provided thatallow regulation of transcription and accumulation of the cognateprotein or nucleotide sequence of interest in fruiting body tissue.Thus, the compositions of the present invention comprise novelnucleotide sequences for fungal regulatory elements natively associatedwith the nucleotide sequences coding for A. bisporus lectin protein.

In an embodiment, the regulatory element drives transcription in afungal tissue or more specifically achieves expression of a protein ortranscript of interest in a fungal fruiting body tissue, wherein saidregulatory element comprises a nucleotide sequence selected from thegroup consisting of: a) sequences natively associated with, and thatregulate expression of DNA coding for lectin protein); b) the sequenceof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or c) asequence comprising a functional fragment of the nucleotide sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

Further embodiments are to expression cassettes, transformation vectors,fungi, bacteria, and bacterial and fungal cells comprising the abovenucleotide sequences. The invention is further to methods of using thesequence in plants, fungi, and bacterial tissues and their cells.

A method for expressing an isolated nucleotide sequence in a fungususing the regulatory sequences disclosed herein is provided. The methodcomprises transforming a fungal cell with a transformation vector thatcomprises an isolated nucleotide sequence operably linked to one or moreof the regulatory sequences of the present invention and regenerating astably transformed fungus from the transformed fungal cell. In thismanner, the regulatory sequences are useful for controlling theexpression of endogenous as well as exogenous products in a fruitingbody tissue.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within the tissues of a fungus to achieve a desiredphenotype. In this case, such inhibition might be accomplished, forexample, with transformation of a fungus to comprise the promoteroperably linked to an antisense nucleotide sequence, hairpin, RNAinterfering or other nucleic acid molecule, such that expression of themolecule interferes with translation of the mRNA of the native DNAsequence or otherwise inhibits expression of same in a subset of thecells of the fungus.

Under the regulation of the regulatory element will be a sequence ofinterest, which will provide for modification of the phenotype of afungus. Such modification includes modulating the production of anendogenous product, as to amount, relative distribution, or the like, orproduction of an exogenous expression product to provide for a novelfunction or product in a fungus. Such a promoter is useful for a varietyof applications, such as production of transgenic fungi with desiredtraits, including, for example, altered content, protein quality, cellgrowth or nutrient quality or preferably for the production ofheterologous proteins.

By “fruiting body tissue” promoter is intended expression that iscapable of transcribing an operatively linked nucleotide sequenceefficiently and thereby achieving accumulation of a protein ornucleotide sequence of interest at high levels in the described tissues,here the fruiting body tissue cells. Tissue can refer to a cell of aparticular tissue.

By “regulatory element” is intended sequences responsible for expressionof the linked nucleic acid molecule including, but not limited to,promoters, terminators, enhancers, introns, and the like.

By “promoter” is intended a regulatory region of DNA capable ofregulating the transcription of a sequence linked thereto. It usuallycomprises a TATA box capable of directing RNA polymerase II to initiateRNA synthesis at the appropriate transcription initiation site for aparticular coding sequence.

A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate and further include elements that impact spatial and temporalexpression of the linked nucleotide sequence. It is recognized thathaving identified the nucleotide sequences for the promoter regiondisclosed herein, it is within the state of the art to isolate andidentify further regulatory elements in the 5′ region upstream from theparticular promoter region. Thus the promoter region disclosed here maycomprise upstream regulatory elements such as those responsible fortissue and temporal expression of the nucleic acid molecule, and mayinclude enhancers, the DNA response element for a transcriptionalregulatory protein, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, activatorsequence and the like.

In the same manner, the promoter elements that enable such expressioncan be identified, isolated, and used with other core promoters toconfirm expression in fruiting body tissue. By core promoter is meantthe minimal sequence required to initiate transcription, such as thesequence called the TATA box, which is common to promoters in genesencoding proteins. Thus the upstream promoter of SB-LEG can optionallybe used in conjunction with its own or core promoters from othersources. The promoter may be native or non-native to the cell in whichit is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the isolated nucleotidesequence. Less than the entire promoter region can be utilized and theability to achieve expression in the fruit body tissue retained. It isrecognized that expression levels of mRNA can be modulated with specificdeletions of portions of the promoter sequence. Thus, the promoter canbe modified to be a weak or strong promoter. Generally, by “weakpromoter” is intended a promoter that drives expression of a codingsequence at a low level. By “low level” is intended levels of about1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts. Generally, at least about 20nucleotides of an isolated promoter sequence will be used to driveexpression of a nucleotide sequence.

It is recognized that to increase transcription levels enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′ UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon.

The term “isolated” refers to material, such as a nucleic acid orprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment, or (2) if the material is in itsnatural environment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art. One method is the use of primers andgenomic DNA used in conjunction with the Genome Walker Kit™ (Clonetech).

The A. bisporus lectin promoter is set forth in SEQ ID NO:1, withfunctional truncations shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The minimal core promoter is 45 nucleotides in length, with thefull-length promoter being approximately 135 to 180 nucleotides inlength. 160 is 818 base pair nucleotides in length. The lectin promoterwas isolated from the A. bisporus lectin coding region.

The regulatory regions of the invention may be isolated from any plantanimal or fungi, but is preferably filamentous fungi, including, but notlimited to the fungi of the genera Flammulina, Agaricus, Lentinula, andPleurotus. Promoters isolated from one fungal species can be expected toexpress in another fungal species.

Regulatory sequences from other fungi may be isolated according towell-known techniques based on their sequence homology to the codingregion of the sequences set forth herein. In these techniques, all orpart of the known coding sequence is used as a probe that selectivelyhybridizes to other sequences present in a population of cloned genomicDNA fragments (i.e. genomic libraries) from a chosen organism. Methodsare readily available in the art for the hybridization of nucleic acidsequences. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

Functional variants of the regulatory sequences are also encompassed bythe compositions of the present invention. Functional variants include,for example, the native regulatory sequences of the invention having oneor more nucleotide substitutions, deletions or insertions. Functionalvariants of the invention may be created by site-directed mutagenesis,induced mutation, or may occur as allelic variants (polymorphisms).

As used herein, a “functional fragment” is a regulatory sequence variantformed by one or more deletions from a larger regulatory element. Forexample, the 5′ portion of a promoter up to the TATA box near thetranscription start site can be deleted without abolishing promoteractivity, as described by Opsahl-Sorteberg et al., “Identification of a49-bp fragment of the HvLTP2 promoter directing aleurone cell specificexpression” Gene 341:49-58 (2004). Such fragments should retain promoteractivity, particularly the ability to drive expression in the selecttissue. Activity can be measured by Northern blot analysis, reporteractivity measurements when using transcriptional fusions, and the like.See, for example, Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′, 4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al. (1990) PCR Protocols, A Guideto Methods and Applications, eds., Academic Press). Primers used inisolating the promoter of the present invention are shown below.

In referring to a terminator sequence is meant a nucleotide sequencethat functions as a polyadenylation signal and signals the end oftranscription. Functional fragments that retain such activity are withinthe scope of the invention.

The regulatory elements disclosed in the present invention, as well asvariants and fragments thereof, are useful in the genetic manipulationof any fungus when operably linked with an isolated nucleotide sequenceof interest whose expression is to be controlled to achieve a desiredphenotypic response.

By “operably linked” is intended a functional linkage between aregulatory region and a second sequence, wherein the regulatory sequenceinitiates and mediates transcription of the DNA sequence correspondingto the second sequence.

The regulatory elements of the invention can be operably linked to theisolated nucleotide sequence of interest in any of several ways known toone of skill in the art. The isolated nucleotide sequence of interestcan be inserted into a site within the genome which is 3′ to thepromoter of the invention using site specific integration as describedin U.S. Pat. No. 6,187,994. The term “nucleotide sequence of interest”refers to a nucleic acid molecule (which may also be referred to as apolynucleotide) which can be an RNA molecule as well as DNA molecule,and can be a molecule that encodes for a desired polypeptide or protein,but also may refer to nucleic acid molecules that do not constitute anentire gene, and which do not necessarily encode a polypeptide orprotein. For example, when used in a homologous recombination process,the promoter may be placed in a construct with a sequence that targetsan area of the chromosome in the fungi but may not encode a protein. Ifdesired, the nucleotide sequence of interest can be optimized for fungitranslation by optimizing the codons used for fungi and the sequencearound the translational start site for fungi. Sequences resulting inpotential mRNA instability can also be avoided.

The regulatory elements of the invention can be operably linked inexpression cassettes along with isolated nucleotide sequences ofinterest for expression in the desired fungi or plant or animal. Such anexpression cassette is provided with a plurality of restriction sitesfor insertion of the nucleotide sequence of interest under thetranscriptional control of the regulatory elements. Alternatively, aspecific result can be achieved by providing for a reduction ofexpression of one or more endogenous products, particularly enzymes orcofactors in fungi. This down regulation can be achieved through manydifferent approaches known to one skilled in the art, includingantisense, co-suppression, use of hairpin formations, or others, anddiscussed infra. Importation or exportation of a cofactor also allowsfor control of composition. It is recognized that the regulatoryelements may be used with their native or other coding sequences toincrease or decrease expression of an operably linked sequence in thetransformed fungi, plant, seed or animal cell.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, and other characteristics. Still other categories oftransgenes include genes for inducing expression of exogenous productssuch as enzymes, cofactors, and hormones. Still others include thoseencoding an antibody, a secondary metabolite, a therapeutic compound, abiological macromolecule, a medical enzyme; or a gene that confers orcontributes to a value-added trait. Examples include a secondarymetabolite such as lectin, a therapeutic compound such as a vaccine, abiological macromolecule such as an interferon, endostatin or insulin, amedical enzyme such as a thrombolytic or cerebrosidase and a gene thatconfers resistance to pests, diseases, or herbicides such as apesticidal compound Bacillus thuringiensis protein (Bt toxin).

It is recognized that any nucleotide sequence of interest, including thenative coding sequence, can be operably linked to the regulatoryelements of the invention and expressed in fungi.

Means for increasing or inhibiting a protein are well known to thoseskilled in the art and, include, but are not limited to: transgenicexpression, antisense suppression, co-suppression, RNA interference,gene activation or suppression using transcription factors and/orrepressors; mutagenesis including, but not limited to, transposontagging; directed and site-specific mutagenesis, chromosome engineering(see Nobrega et. al., Nature 431:988-993(04)), homologous recombination,TILLING (Targeting Induced Local Lesions In Genomes), and biosyntheticcompetition to manipulate the expression of proteins. Many techniquesfor gene silencing are well known to one of skill in the art, includingbut not limited to knock-outs (such as by insertion of a transposableelement such as Mu, Vicki Chandler, The Maize Handbook ch. 118(Springer-Verlag 1994) or other genetic elements such as a FRT, Lox orother site specific integration site; RNA interference (Napoli et al.(1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323, Sharp (1999) GenesDev. 13:139-141, Zamore et al. (2000) Cell 101:25-33; and Montgomery etal. (1998) PNAS USA 95:15502-15507); virus-induced gene silencing(Burton et al. (2000) Plant Cell 12:691-705, and Baulcombe (1999) Curr.Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff etal. (1988) Nature 334:585-591); hairpin structures (Smith et al. (2000)Nature 407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman &Sakai (2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al.(1992) EMBO J. 11:1525, and Perriman et al. (1993) Antisense Res. Dev.3:253); oligonucleotide mediated targeted modification (e.g., WO03/076574 and WO 99/25853); zinc-finger targeted molecules (e.g., WO01/52620; WO 03/048345; and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. (See,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453, 566; and 5,759,829). By “antisense DNA nucleotidesequence” is intended a sequence that is in inverse orientation to the5′-to-3′ normal orientation of that nucleotide sequence. When deliveredinto a cell, expression of the antisense DNA sequence prevents normalexpression of the DNA nucleotide sequence for the targeted gene. Theantisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing with the endogenousmessenger RNA (mRNA) produced by transcription of the DNA nucleotidesequence for the targeted gene. In this case, production of the nativeprotein encoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the regulatory sequences disclosed herein canbe operably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in fungi.

As noted, other potential approaches to impact expression of proteins inthe fungi include traditional co-suppression, that is, inhibition ofexpression of an endogenous gene through the expression of an identicalstructural gene or gene fragment introduced through transformation(Goring, D. R., Thomson, L., Rothstein, S. J. 1991. Proc. Natl. Acad.Sci. USA 88:1770-1774 co-suppression; Taylor (1997) Plant Cell 9:1245;Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS USA91:3490-3496; Finnegan et al. (1994) Bio/Technology 12:883-888; andNeuhuber et al. (1994) Mol. Gen. Genet. 244:230-241)). In one example,co-suppression can be achieved by linking the promoter to a DNA segmentsuch that transcripts of the segment are produced in the senseorientation and where the transcripts have at least 65% sequenceidentity to transcripts of the endogenous gene of interest, therebysuppressing expression of the endogenous gene in said fungal cell. (See,U.S. Pat. No. 5,283,184). The endogenous gene targeted forco-suppression may be a gene encoding any protein that accumulates inthe fungal species of interest.

Additional methods of down-regulation are known in the art and can besimilarly applied to the instant invention. These methods involve thesilencing of a targeted gene by spliced hairpin RNA's and similarmethods also called RNA interference and promoter silencing (see Smithet al. (2000) Nature 407:319-320, Waterhouse and Helliwell (2003)) Nat.Rev. Genet. 4:29-38; Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA95:13959-13964; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731;and Patent Application WO 99/53050; WO 99/49029; WO 99/61631; WO00/49035 and U.S. Pat. No. 6,506,559.

For mRNA interference, the expression cassette is designed to express anRNA molecule that is modeled on an endogenous miRNA gene. The miRNA geneencodes an RNA that forms a hairpin structure containing a 22-nucleotidesequence that is complementary to another endogenous gene (targetsequence). miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of fungi.

The regulatory region of the invention may also be used in conjunctionwith another promoter. In one embodiment, the fungal selection markerand the gene of interest can be both functionally linked to the samepromoter. In another embodiment, the selection marker and the gene ofinterest can be functionally linked to different promoters. In yet otherembodiments, the expression vector can contain two or more genes ofinterest that can be linked to the same promoter or different promoters.For example, the SB-LEG promoter described here can be used to drive thegene of interest and the selectable marker, or a different promoter usedfor one or the other. These other promoter elements can be those thatare constitutive or sufficient to render promoter-dependent geneexpression controllable as being cell-type specific, tissue-specific ortime or developmental stage specific, or being inducible by externalsignals or agents. Such elements may be located in the 5′ or 3′ regionsof the gene. Although the additional promoter may be the endogenouspromoter of a structural gene of interest, the promoter can also be aforeign regulatory sequence. Promoter elements employed to controlexpression of product proteins and the selection gene can be anycompatible promoter. These can be fungal gene promoters, such as, forexample, gpd promoter, or promoters from the tumor-inducing plasmidsfrom Agrobacterium tumefaciens, such as the nopaline synthase, octopinesynthase and mannopine synthase promoters (Velten, J. and Schell, J.(1985) “Selection-expression plasmid vectors for use in genetictransformation of higher plants” Nucleic Acids Res. 13:6981-6998;Depicker et al., (1982) Mol. and Appl. Genet. 1:561-573 Shaw et al.(1984) Nucleic Acids Research vol. 12, No. 20 pp. 7831-7846) that havefungal activity; or viral promoters such as the Cauliflower mosaic virus(CaMV) 19S and 35S promoters (Guilley et al. (1982) “Transcription ofCauliflower mosaic virus DNA: detection of promoter sequences, andcharacterization of transcripts” Cell 30:763-773; Odell et al. (1985)“Identification of DNA sequences required for activity of thecauliflower mosaic virus 35S promoter” Nature 313:810-812, the figwortmosaic virus FLt promoter (Maiti et al. (1997) “Promoter/leader deletionanalysis and plant expression vectors with the figwort mosaic virus(FMV) full length transcript (FLt) promoter containing single or doubleenhancer domains” Transgenic Res. 6:143-156) or the coat proteinpromoter of TMV (Grdzelishvili et al., 2000) “Mapping of the tobaccomosaic virus movement protein and coat protein subgenomic RNA promotersin vivo” Virology 275:177-192).

The expression cassette may also include at the 3′ terminus of theisolated nucleotide sequence of interest, a transcriptional andtranslational termination region functional in fungi. The terminationregion can be native with the promoter nucleotide sequence of thepresent invention, can be native with the DNA sequence of interest, orcan be derived from another source. Thus, any convenient terminationregions can be used in conjunction with the promoter of the invention,and are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also:Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987)Nucleic Acid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco etch virus), Allisonet al. (1986); MDMV leader (Maize dwarf mosaic virus), Virology154:9-20; human immunoglobulin heavy-chain binding protein (BiP),Macejak et al. (1991) Nature 353:90-94; untranslated leader from thecoat protein mRNA of Alfalfa mosaic virus (AMV RNA 4), Jobling et al.(1987) Nature 325:622-625); Tobacco mosaic virus leader (TMV), Gallie etal. (1989) Molecular Biology of RNA, pages 237-256; and Maize chloroticmottle virus leader (MCMV), Lommel et al. (1991) Virology 81:382-385.See also Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have an expressed product ofan isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions, such astransitions and transversions, can be involved.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample: Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al.(1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J.9:2517-2522; Kain et al. (1995) Bio Techniques 19:650-655; and Chiu etal. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to:genes encoding resistance to chloramphenicol, Herrera Estrella et al.(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella et al. (1983)Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian etal. (1995) Plant Science 108:219-227; streptomycin, Jones et al. (1987)Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard et al.(1996) Transgenic Res. 5:131-137; bleomycin, Hille et al. (1990) PlantMol. Biol. 7:171-176; sulfonamide, Guerineau et al. (1990) Plant Mol.Biol. 15:127-136; bromoxynil, Stalker et al. (1988) Science 242:419-423;glyphosate, Shaw et al. (1986) Science 233:478-481; phosphinothricin,DeBlock et al. (1987) EMBO J. 6:2513-2518 including the maize optimized“pat” gene, Gordon-Kamm (1990) The Plant Cell 2: 603; Uchimiya et al.(1993) Bio/Technology 11: 835; and Anzai et al. (1989) Mol. Gen. Gen.219:492).

Further, when linking a promoter of the invention with a nucleotidesequence encoding a detectable protein, expression of a linked sequencecan be tracked in fungi, thereby providing useful screenable or scorablemarkers. The expression of the linked protein can be detected withoutthe necessity of destroying tissue. By way of example withoutlimitation, the promoter can be linked with detectable markers includinga β-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (Jefferson et al., 1986, Proc.Natl. Acad. Sci. USA 83:8447-8451); chloramphenicol acetyl transferase;alkaline phosphatase; a R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in tissues(Dellaporta et al., in Chromosome Structure and Function, KluwerAcademic Publishers, Appels and Gustafson eds., pp. 263-282 (1988);Ludwig et al. (1990) Science 247:449); a p-lactamase gene (Sutcliffe,Proc. Nat'l. Acad. Sci. U.S.A. 75:3737 (1978)), which encodes an enzymefor which various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l.Acad. Sci. U.S.A. 80:1101 (1983)), which encodes a catechol dioxygenasethat can convert chromogenic catechols; an α-amylase gene (Ikuta et al.,Biotech. 8:241 (1990)); a tyrosinase gene (Katz et al., J. Gen.Microbiol. 129:2703 (1983)), which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone, which in turn condenses toform the easily detectable compound melanin a green fluorescent protein(GFP) gene (Sheen et al., Plant J. 8(5):777-84 (1995)); a lux gene,which encodes a luciferase, the presence of which may be detected using,for example, X-ray film, scintillation counting, fluorescentspectrophotometry, low-light video cameras, photon counting cameras ormultiwell luminometry (Teeri et al. (1989) EMBO J. 8:343); DS-REDEXPRESS (Matz et al. (1999) Nature Biotech. 17:969-973, Bevis et al.(2002) Nature Biotech 20:83-87, Haas et al. (1996) Curr. Biol.6:315-324); Zoanthus sp. yellow fluorescent protein (ZsYellow) that hasbeen engineered for brighter fluorescence (Matz et al. (1999) NatureBiotech. 17:969-973, available from BD Biosciences Clontech, Palo Alto,Calif., USA, catalog no. 632443); and cyan florescent protein (CYP)(Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002)Plant Physiol 129:913-42).

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in fungal cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleicacids). Vectors and procedures for cloning and expression in E. coli arediscussed in Sambrook et al. (supra). The transformation vectorcomprising the regulatory sequence of the present invention operablylinked to an isolated nucleotide sequence in an expression cassette, canalso contain at least one additional nucleotide sequence for a gene tobe co-transformed into the organism. Alternatively, the additionalsequence(s) can be provided on another transformation vector. Vectorsthat are functional in fungi can be binary plasmids derived fromAgrobacterium. Such vectors are capable of transforming fungal cells.These vectors contain left and right border sequences that are requiredfor integration into the host chromosome. At minimum, between theseborder sequences is the gene to be expressed under control of theregulatory elements of the present invention. In one embodiment, aselectable marker and a reporter gene are also included.

A transformation vector comprising the particular regulatory sequencesof the present invention, operably linked to an isolated nucleotidesequence of interest in an expression cassette, can be used to transformany fungi. In this manner, genetically modified fungi, fungal cells,fungal tissue, and the like can be obtained. Transformation protocolscan vary depending on the type of cell. Suitable methods of transformingfungal cells include microinjection, Crossway et al. (1986)Biotechniques 4:320-334; electroporation, Riggs et al. (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606; Agrobacterium-mediatedtransformation, see for example, Townsend et al. U.S. Pat. No.5,563,055; Romaine et al U.S. Pat. No. 7,700,439, direct gene transfer,Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particleacceleration, see for example, Sanford et al. U.S. Pat. No. 4,945,050.

The cells that have been transformed can be grown into fungi inaccordance with conventional methods. These fungi can then be reproducedwith the same transformed strain or different strains. The resultingfungi having constitutive expression of the desired phenotypiccharacteristic can then be identified. Two or more generations can begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited.

The following examples are intended to further illustrate the inventionand are not to limit the invention in any way. The examples anddiscussion herein may specifically reference A. bisporus, however theteachings herein are equally applicable to any other fungus, preferablyfilamentous fungi that bear fleshy fruit bodies.

EXAMPLES Example 1 Identification of a Highly Expressed Lectin GeneSequence from Agaricus bisporus

Using 2-D protein profiling of total soluble protein, a lectin gene wasdetermined to be highly expressed in the fruiting body of A. bisporus.Fruiting bodies (commercial intermediate hybrid strain) selected werenear maturity, but without exposed gills. Freshly harvested fruit tissuewas frozen in liquid nitrogen, pulverized using a mortar and pestle, andextracted with cold acetone with 20% trichloroacetate and 0.2%dithiothreitol and submitted to Alphalyse Inc. (Palo Alto, Calif.) for2-D protein profiling. The resulting gel image is presented in FIG. 1.

FIG. 1. 2-D Gel Analysis of Agaricus bisporus Fruiting Body Proteins.

Protein separation on 2-D SDS PAGE was performed according to themanufacturer's recommendation using IPG drystrips for the firstdimension isoelectric focusing, and NuPAGE pre-cast gel system fromInvitrogen for the 2nd dimension separation. The numbers identifyprotein spots excised for MS and MS/MS analysis.

Following electrophoresis, several proteins were identified as highlyabundant based on staining intensity on the 2-D gel image. Proteins #4,#6 and #7 (FIG. 1) were among the most abundant and, therefore, werechosen for additional analysis, including peptide mapping and sequencinganalysis carried out by Alphalyse Inc. The resulting peptide map andpartial peptide sequence information of proteins #4, #6, and #7 wereblast searched against a protein database created by Alphalyse Inc. andwere shown to match the cDNA sequence of the A. bisporus lectin gene(U14936, Crenshaw et al. 1996). The three proteins shared an identicalamino acid sequence. The observed separation of the lectin protein intothree distinct protein spots probably reflects variation inpost-translational modifications, such as glycosylation.

Example 2 Isolation and Sequence Determination of the 5′ Upstream DNARegion of the Lectin Gene Encompassing the Promoter

The isolation of the genomic 5′ upstream DNA region of the lectin genewas carried out using genome walking (APAgene™ Gold Genome Walking Kit,BIO S&T Inc. Montreal, QC, Canada). The following primers: lectin-F1(5′-TTCGTTCAACGGGACGGAAGAAGCCTTT-3′) and lectin-F2(5′-TCTGGTAGACGCGAATGCTGATGGTGTA-3′) were designed from the lectin cDNAsequence (Crenshaw et al., 1995) and used for genome walking Genomic DNAof A. bisporus was extracted using an AquaGenomic Kit (MultiTargetPharmaceuticals LLC, Salt Lake City, Utah) and used as the template forgenome-walking PCR according to the protocol provided with the APAgene™Gold Genome Walking Kit. Briefly, four 15-μl aliquots of the PCRreaction mixture were added to PCR tubes labeled A, B, C, and D, using3×APAgene Gold buffer I for tubes A and B, and APAgene™ Gold buffer IIfor tubes C and D. The 15 μl of primary PCR mixture contained 5 μl of3×APAgene™ Gold buffer, 0.3 μl of 50×PCR annealing enhancer, 0.4 μl of40 mM dNTPs, 0.7 μl of 20 pmol/μl of lectin-F1, 1 μl of genomic DNA(−100 ng), 0.2 μl (1 U) of Taq DNA polymerase (Platinum Taq, Invitrogen,Carlsbad, Calif.) and 7.4 μl of water. The PCR program consisted of onecycle at 94° C. for 4 min followed by 25 cycles at 94° C. for 30 sec,63° C. for 10 sec, ramp to 66° C. at 0.1° C./sec, and 68° C. for 3 min.and a final cycle at 68° C. for 10 min. Following PCR amplification, 0.3μl (1.5 U) of Taq polymerase was added to each tube, and 1 μl of 15×degenerate random tagging (DRT) primer A, B, C and D to tube A, B, C andD, respectively. Reaction mixtures were vortexed and subjected to asecond PCR program consisting of one cycle at 94° C. for 2 min followedby 1 cycle at 94° C. for 30 sec, 25° C. for 10 sec, ramp to 65° C. at0.1° C./sec, 68° C. for 6 min, then 20 cycles of 94° C. for 30 sec, 55°C. for 30 sec, 68° C. for 2 min and 50 sec, and a final cycle at 68° C.for 30 min. Following the secondary PCR amplification, 0.5 μl of the PCRproduct from A, B, C and D tubes was transferred to tubes labeled A1,B1, C1, and D1, respectively. A 1 μl aliquot of 10×DRT primer digestionbuffer was added to each tube with 0.5 μl of digestion enzyme mix, and 8μl of water. The digestion mixtures were vortexed and incubated at 37°C. for 30 min. The enzyme in the digestion mixtures was thenheat-inactivated at 95° C. for 5 min. Following the digestion reactionsthe 5′ genomic DNA region upstream of the lectin gene was amplified bynested PCR. For nested PCR, 15 μl of reaction mixture were added to eachof four tubes labeled A2, B2, C2, and D2, using 3×APAgene™ Gold buffer Ifor tubes A2 and B2, and APAgene™ Gold buffer II for tubes C2 and D2.The 15 μl of reaction mixture for the nested PCR contained 5 μl of3×APAgene™ Gold buffer (I or II), 0.3 μl of 50×PCR annealing enhancer,0.3 μl of 40 mM dNTPs, 0.4 μl of 20 pmol/μl of lectin-F2 primer, 0.4 μlof Universal Tagging Primer, 0.5 μl of digested PCR product from tubesA1, B1, C1, or D1, and 0.3 μl of Taq DNA polymerase (Invitrogen) and 7.8μl of water. The nested PCR program for amplification of the putativelectin genes consisted of one cycle at 94° C. for 4 min followed by 30cycles at 94° C. for 30 sec, 62° C. for 10 sec, ramp to 65° C. at 0.1°C./sec, followed by 68° C. for 2 min and 30 sec and a final cycle at 68°C. for 10 min. PCR products were separated in a 1% agarose gel in 1×TBE(1×TRIS/BORATE/EDTA, 54 g of Tris base, 27.5 g of boric acid, 20 ml of0.5 M EDTA (pH 8.0) in 1000 ml) by gel electrophoresis. The PCR productsof reactions A2, C2 and D2 ranged in size from ˜400 bp to ˜600 bp.Amplicons were recovered from the agarose gel using a Quick GelExtraction Kit (Invitrogen).

The PCR amplification products of the putative 5′ genomic DNA regionsupstream of the lectin genes were cloned into a pCR2.1 TOPO vector usinga TOPO TA Cloning Kit (Invitrogen) following the manufacturer'sprotocol. Briefly, 4 μl of PCR product recovered from the agarose gelwas added to 6 μl of pCR2.1 ligation reaction. After ligation for 5 minat room temperature, 3 μl of each reaction mixture were used totransform ONE-SHOT DH5α chemical competent cells (Invitrogen). Thetransformed cells were plated and selected on LB (Growcells, Irvine,Calif.) agar plate containing 50 μg/ml of kanamycin with 40 μl of 40mg/ml of X-gal (MP Biomedicals, Solon, Ohio) spread on the surface.After overnight incubation at 37° C. in the dark, white colonies werepicked for plasmid DNA preparation. Plasmid DNA was digested with EcoRIand separated by 1% agarose gel electrophoresis in 1×TBE (describedabove) to identify clones with the correct insert sizes. The clonescontaining PCR product from the A2 reaction were designated pAGN-743,those from the C2 reaction as pAGN-744 and those from D2 as pAGN-745.

The nucleotide sequence of the lectin gene promoter was determined bysequencing the inserts in pAGN-743, pAGN-744 and pAGN745 using anM13R-reverse primer. Alignment among the insert sequences of pAGN-743,pAGN-744 and pAGN-745 clones revealed that the three PCR productsoverlapped. pAGN745 clones (pAGN-745-1, -2 and -3) contained the longest5′ genomic DNA region upstream of the lectin gene. The consensussequence of the 5′ genomic DNA region upstream of the lectin gene wasgenerated using sequence information from pAGN-745 clones. The overlapof the consensus sequence with the 5′ end cDNA sequence of lectin geneconfirmed that the DNA fragment generated by genome walking representedthe promoter region of the lectin gene. Alignment between the genomicDNA sequence generated by genome walking and the lectin cDNA sequence(U14936, Crenshaw et al. 1996) revealed an intron in the 5′ UTR oflectin mRNA. A nucleotide blast search (blastn) of the consensussequence of the 5′ genomic DNA region upstream of the lectin geneagainst “all GenBank+EMBL+DDBJ+PDB sequences” failed to show identitywith any known sequences, indicating that the isolated sequence was anovel promoter.

Example 3 Demonstrated Ability of the Lectin Promoter to DirectExpression of the GUS Reporter Gene in Agaricus bisporus

Binary vector pAGN-750 (FIG. 2) was constructed to test the ability ofthe lectin promoter (designated as pLctn) to drive the expression of aβ-glucuronidase (GUS) reporter gene in A. bisporus. pLctn was amplifiedfrom pAGN-745 using primers Lctn-BglII (5′-AGCTTAGATCTGAACACGCGTCGTTTACCTCC-3′) and Lctn-NcoI (5′-GTAAGTCCATGGTCTGCTCAACGTTATGAGTTAGTTG-3′). Restriction enzyme sites, BglII and NcoI, were incorporatedinto primers to facilitate cloning. The PCR product was digested withBglII/NcoI and the digested product was used to replace the BglII-NcoIfragment in GUS expression vector pAGN-030 to create pAGN-750. The pLctnsequence in pAGN-750 was confirmed by sequencing. pAGN-750 wasintroduced into Agrobacterium tumefaciens and then A. bisporus followingthe transformation procedure described by Chen et al. (2000). Transgenicevents generated from pAGN-750 were analyzed for GUS expression (GUSprotocol by Sean R. Gallagher, 1991) in the vegetative mycelium andreproductive fruiting body. For determining GUS expression in themycelium, mycelium of transgenic events was allowed to colonize filterpaper for 10-14 days on SM agar plate containing 30 μg/ml of hygromycinB. GUS expression in mycelium was evaluated based on the intensity ofblue color present in the mycelial colony exposed to staining buffer onfilter paper (FIG. 3). The results of this study showed that pLctn droveGUS expression in the vegetative growth stage of A. bisporus. GUSexpression in fruit tissue was also evaluated. Mycelia of transgenicevents were used to inoculate growth media for mushroom production.Following transformation and selection of transgenic lines onhygromycin, cultures were transferred to liquid media (2% malt extractbroth (Difco) and maintained there at room temperature pendinginoculation of fruiting media. The liquid media culture was mixed with asterilized rye/millet grain mixture (50:50) in sterilized 250 ml plasticcontainers and the transgenic A. bisporus mycelia was allowed tocolonize the grain substrate for approximately two weeks at roomtemperature. The fully colonized grain was overlaid with a three cmdepth of peat humus casing mixture and the cultures were transferred to18° C. incubators when mycelium emerged on the top of casing. Cultureswere watered frequently to maintain the moist growing environmentpreferred by the organism. Mature fruit was harvested prior tosporulation and either placed immediately at −80° C. for storage andfuture analysis or for gus expressing lines, stored briefly at 4° C.prior to analysis by GUS visual staining or the MUG quantitative assay.

For visualization of gus gene expression, 3-mm thick slice from eachfruiting body was submerged in GUS staining buffer. GUS expression infruiting bodies was recorded according to a visual estimation of theintensity of color development in the tissues (Table 1). The findings ofthis study (Table 1, FIG. 3) showed that pLctn can direct high-level GUSaccumulation in the reproductive fruiting body of A. bisporus. Variationin the levels of staining intensity among different independenttransgenic events was observed for the pAGN-750 construct. Thisvariation can be explained by differences in sequence context resultingfrom integration of the transgene into different chromosomal locationsand/or by differences in transgene copy number between events, which iscommonly observed for other promoter/transgene combinations transformedinto A. bisporus and routinely reported in other plant, animal andfungal systems (Peach C. and Velten J. 1991; Siegal M. L. and Hartl D.L. 1998; Butaye et al. 2005). Fruiting bodies from five events werechosen to quantitatively measure GUS activity using the MUG(4-Methylumbelliferyl glucuronide) assay (Gallagher, S. R. 1991). Toextract total soluble protein for the MUG assay, 100 mg of fruiting bodytissue were added to a tube containing 300 ul of PBS buffer (1×PBS: 8 gNaCl, 0.2 g KCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄ in 1000 ml, pH 7.4)with 3 glass beads (3.5 mm in diameter) and homogenized at 400strikes/min for 3 min with Geno/Grinder (SPEX SamplePrep 2000Geno/Grinder, SPEX SamplePrep LLC, Metuchen, N.J.). Proteinconcentration in the soluble extract was quantified following themanufacturer's instruction for the BCA Protein Assay Kit (ThermosScientific, Rockford, Ill.). Quantification of GUS activity in fruitingbody tissue is presented as MUG reading per μg of total soluble proteinextracted from the tissues. The MUG assay data (Table 2) confirmed theresults of the qualitative analysis of GUS expression in the fruitingbody. The results are shown in FIG. 3.

TABLE 1 Visual scoring of GUS accumulation in fruiting body tissue GusExpression Event* Level** 750-1A 0.5 750-2A 0.5 750-3A 3.5 750-4A 2750-5A 2 750-6A 1.5 750-7A 3 750-8A 4 750-9A 2.5 750-10A 1 750-11C 4750-12A 2.5 750-13A 2.5 750-14A 2.5 750-15B 0.5 750-16A 3.5 750-17A 0.5750-18B 2 750-19B 4 750-20C 2 750-21A 1.5 750-22A 4 750-23A 1.5 750-24A4 *A, B and C represent fruiting body A, B, and C, respectively, of theevent **Fruiting body slices were submerged in GUS staining buffer for 6hours at RT and the enzyme reaction was stopped by two rinses in waterand transfer to a 50% ethanol solution. Rating scale: 1 = pale blue; 2 =moderate blue; 3 = dark blue; 4 = deep dark blue

TABLE 2 Quantitative measurement of GUS activity in the fruiting body.MUG/Total Protein Ratio for Protein Samples of Whole Fruiting BodySection Event ID 750- 750- 750- 750- 750- 3A 8A 16A 19B 22A Average Mugreading/ug 24.7 85.1 79.1 153.2 130.9 93.4 total protein* *MUG reactionwas terminated by the addition of 150 ul of NaCO₂ after 15 min at 37 C..FIG. 4. GUS Staining of Fruiting Body Slices for Independent TransgenicEvents of pAGN-750.Sample 1=750-22B; sample 2=750-16A; sample 3=750-6B; sample 4=750-11C;sample 5=750-7A; sample 6=750-13A; sample 7=750-8A; sample 8=750-23A

Example 4 Demonstrated Ability of the Lectin Promoter to DirectExpression of Bovine Trypsin Inhibitor (Aprotinin) in Agaricus bisporus

Binary vector pAGN-755 (FIG. 5) was constructed to test the ability ofpLctn to effect expression of an Aprotinin target gene. ConstructpAGN-755 was synthesized by replacing the BglII-NcoI promoter fragmentin aprotinin expression vector pAGN-021 using the BglII-NcoI-pLctnfragment released from pAGN-750. The pLctn sequence in pAGN-755 wasconfirmed by sequencing. pAGN-755 was introduced into A. tumefaciens andthen A. bisporus after the transformation procedure described by Chen etal. (2000). Fruiting bodies were produced as described previously for apanel of transgenic events generated using pAGN-755. Aprotininexpression in fruiting body tissue was analyzed using an enzyme-linkedimmunosorbent assay (ELISA) and Western blotting. High expression ofaprotinin, up to 200 mg/kg of fresh fruiting body tissue, was observed(Table 3, FIG. 4).

TABLE 3 Aprotinin expression in pAGN-755 events Representative EventsExpression (mg/kg) 755-B10-304 14.20 755-B10-314 54.69 755-B10-325 36.40755-B10-332 35.37 755-B10-348 24.54 755-B10-352 17.27 755-B10-354 15.13755-B10-360 42.99 755-B10-363A 139.65 755-B10-363B 205.32 755-B10-36411.33 755-B10-380 129.22 755-B10-381 22.05 755-B10-386 26.78 755-B10-3888.21 755-B10-394 18.96

Example 5 Determination of the DNA Sequence Essential for LectinPromoter Activity

The minimal region of the 5′ genomic DNA region upstream of the lectingene required for promoter activity was defined by deletion analysis.The following PCR primers were designed and synthesized in order toevaluate the capability of individual segments of this sequence toeffect gene expression:Lctn-BglII-2,5′-AGCTTAGATCTGTCCTGGCACTAGTCACAG-3′,Lctn-BglII-4,5′-AGCTTAGATCTTTGTAGCGCTTGATGC-3′,Lctn-BglII-6,5′-AGCTTAGATCTTATAATTAGCACATACCC-3′,Lctn-BglII-7,5′-AGCTTAGATCT CGTCTTCATAGATATGGCCAAGCATC-3′, Lctn-BglII-8,5′-AGCTTAGATCT TGCTACTTGTAGATAAGTTTATCTACACC-3′, Lctn-BglII-9,5′-AGCTTAGATCTGATATATAACGATGTCCGATACTTGTG-3′, and Lctn-BglII-10,5′-AGCTTAGATCTCTTGACTGCGATTCACCTGCTCC-3′. Primer pairs used for promoteranalysis and the expected sizes are listed in Table 4. BglII-NcoIdigested PCR products were used to replace the full-length 5′ genomicDNA region upstream of the lectin gene in pAGN-774. The resultingvectors were designated pAGN-1279, pAGN-1280, pAGN-1281, pAGN-1282,pAGN-1283, pAGN-1304, and pAGN-1305, as described in Table 4 and FIG. 6.

TABLE 4 Primers and expected promoter size for pLctn deletion analysisExpected Length Gus Primer A Primer B of Promoter Vector ExpressionLctn-BglII-2 Lctn-NcoI 474 bp pAGN-1279 Strong Lctn-BglII-4 Lctn-NcoI369 bp pAGN-1280 Strong Lctn-BglII-6 Lctn-NcoI 280 bp pAGN-1281 StrongLctn-BglII-7 Lctn-NcoI 230 bp pAGN-1282 Strong Lctn-BglII-8 Lctn-NcoI180 bp pAGN-1283 Strong Lctn-BglII-9 Lctn-NcoI 135 bp pAGN-1304 WeakLctn-BglII-10 Lctn-NcoI  85 bp pAGN-1305 WeakFIG. 6 shows the Lectin promoter sequences evaluated. 5′ ends DNAsequence in each vector are underlined and the corresponding vectoridentified. All vectors share the same 3′ sequence presented. pAGN-755and pAGN-750 share the identical sequence.

The constructs incorporating fragments of the 5′ region of the lectingene were transformed into A. tumefaciens and then A. bisporus asdescribed previously. GUS expression driven by the three truncatedpromoter sequences was compared to that obtained with the full-length 5′genomic DNA region upstream of the lectin gene present in pAGN-774. Thesequences are shown in Table 5.

TABLE 5 DNA sequences in each vector used in lectin pro- moter analysisVectors Promoter sequence pAGN-GATCTGAACACGCGTCGTTTACCTCCGGGGTGAGTCTCCTGG 750 andCACCTTGACAGAATCTAGAATACGATGATCGCACCCTCGATT pAGN-CCCATGAGCTAAAAATATCTGGTCCTGGCACTAGTCACAGTG 755GTCACCGACTCGAAAATTTCCCCGTCCATAGTTAACTTTTTTCAACCACAAGTACTCAATCAAATCTACTAGCTTGTAGAAACATTGTAGCGCTTGATGCTGAATAAATTCAATGCAATCATATAGCATACTACAGATCAACAGGTGCTCAAGCTCAAGCGGGACACTCGATCTATAATTAGCACATACCCCAAAGTGCGGGGGTTAATGGCGCTGGACACTTCGTCTTCATAGATATGGCCAAGCATCCTTCATGCCATGCTCGAATTATGCTACTTGTAGATAAGTTTATCTACACCCGCACGGACCAACTAACGTAGATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGATTATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGTTTCAACTAACTCATAACGTTGAGCAGACC pAGN-GTCCTGGCACTAGTCACAGTGGTCACCGACTCGAAAATTTCC 1279CCGTCCATAGTTAACTTTTTTCAACCACAAGTACTCAATCAAATCTACTAGCTTGTAGAAACATTGTAGCGCTTGATGCTGAATAAATTCAATGCAATCATATAGCATACTACAGATCAACAGGTGCTCAAGCTCAAGCGGGACACTCGATCTATAATTAGCACATACCCCAAAGTGCGGGGGTTAATGGCGCTGGACACTTCGTCTTCATAGATATGGCCAAGCATCCTTCATGCCATGCTCGAATTATGCTACTTGTAGATAAGTTTATCTACACCCGCACGGACCAACTAACGTAGATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGATTATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGTTTCAACTAACTCATAA CGTTGAGCAGACC pAGN-TTGTAGCGCTTGATGCTGAATAAATTCAATGCAATCATATAG 1280CATACTACAGATCAACAGGTGCTCAAGCTCAAGCGGGACACTCGATCTATAATTAGCACATACCCCAAAGTGCGGGGGTTAATGGCGCTGGACACTTCGTCTTCATAGATATGGCCAAGCATCCTTCATGCCATGCTCGAATTATGCTACTTGTAGATAAGTTTATCTACACCCGCACGGACCAACTAACGTAGATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGATTATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGTTTCAACTAACTCATAACGTTGAGCAGACC pAGN-TATAATTAGCACATACCCCAAAGTGCGGGGGTTAATGGCGCT 1281GGACACTTCGTCTTCATAGATATGGCCAAGCATCCTTCATGCCATGCTCGAATTATGCTACTTGTAGATAAGTTTATCTACACCCGCACGGACCAACTAACGTAGATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGATTATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGT TTCAACTAACTCATAACGTTGAGCAGACCpAGN- CGTCTTCATAGATATGGCCAAGCATCCTTCATGCCATGCTCG 1282AATTATGCTACTTGTAGATAAGTTTATCTACACCCGCACGGACCAACTAACGTAGATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGATTATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGTTTCAACTA ACTCATAACGTTGAGCAGACC pAGN-TGCTACTTGTAGATAAGTTTATCTACACCCGCACGGACCAAC 1283TAACGTAGATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGATTATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGTTTCAACTAACTCA TAACGTTGAGCAGACC pAGN-GATATATAACGATGTCCGATACTTGTGAATACCTCAGTGGAT 1304TATATCCTCTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCTTTCTCTACCACTGTTTCAACTAACTCATAACGTT GAGCAGACC pAGN-CTTGACTGCGATTCACCTGCTCCACTAACACAGTAACCATCT 1305TTCTCTACCACTGTTTCAACTAACTCATAACGTTGAGCAGACCFIG. 7 shows mycelium GUS staining of the events generated from thetruncated promoters. Mushroom fruit bodies generated from pAGN-1304 andpAGN-1305 also show very weak or no GUS expression in GUS staining (datanot shown).Data suggested that the minimal full-strength of lectin promoter isbetween 135 bp to 180 bp, as demonstrated by pAGN-1283, pAGN-1304 andpAGN-1305 GUS expression data.

REFERENCES

-   Anne-Marie Stomp. Histochemical localization of b-Glucuronidase. pps    103-124 In GUS protocols: using the GUS gene as a reporter of gene    expression. 1991 Edited by Sean R. Gallagher.-   Chen, X., Stone, M., Schlagnhaufer, C., Romaine, C. P., 2000. A    fruiting body tissue method for efficient Agrobacterium-mediated    transformation of Agaricus bisporus. Appl. Environ. Microbiol. 66,    4510-4513.-   Crenshaw, R. W., Harper, S. N., Moyer, M. and Privalle, L. S. (1995)    Isolation and characterization of a cDNA clone encoding a lectin    gene from Agaricus bisporus. Plant Physiol. 107(4):1465-1466 (1995).-   Rao, G. A. and Flynn, P. Microtiter plate-based assay for    b-D-Glucuronidase: a quantitative approach. pp. 89-99. In GUS    protocols: using the GUS gene as a reporter of gene expression. 1991    Edited by Sean R. Gallagher.-   Peach, C. and Velten, J. (1991) Transgene expression variability    (position effect) of CAT and GUS reporter genes driven by linked    divergent T-DNA promoters. Plant Molecular Biology vol. 17:49-60.-   Butaye, K. M. J., Cammue, B. P. A., Delaure S. L., and De    Bolle M. F. C. 2005 Approaches to minimize variation of transgene    expression in plants. Molecular Breeding Vol. 16:79-91.-   Siegal, M. L. and Hartl, D. L. (1998) An experimental test for    lineage-specific position effects on alcohol dehydrogenase (Adh)    genes in Drosophila Proc. Natl. Acad. Sci. USA Vol. 95, 15513-15518.

Media Selection Medium (SM) Agar

500 ml   10 g malt extract 1.05 g MOPS (or 1.15 MOPS sodium salt) 1.5%agar (7.5 g) Add water to 500 ml pH to 7.0 & BTV Autoclave

Selection Medium (SM)

500 ml   10 g Malt extract 1.05 g MOPS (or 1.15 MOPS sodium salt) 1.5%agar (7.5 g) Add water to 500 ml pH to 7.0 & BTV Autoclave   50 μg/mlHygromycin B (500 μl of 50 mg/ml)  200 μM Cefotaxim (500 μl of 100mg/ml)  100 μg/ml Moxalactum (optional)

GUS Stain Solution

100 ml   2 ml 0.5M EDTA (pH 8.0) 1.38 g NaH₂PO₄  100 μl Triton X-100 Addwater to 100 ml pH to 7.0 with NaOH 0.05 g x-gluc in 2 ml DMF

What is claimed is:
 1. An isolated regulatory element that drivesaccumulation of either a protein or nucleotide sequence of interest infruiting body tissue, wherein the regulatory element comprises thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:4.
 2. An expression cassette comprising the regulatory element ofclaim 1 wherein said regulatory element is operably linked to anucleotide sequence.
 3. A fungus stably transformed with the expressioncassette of claim
 2. 4. The fungus of claim 3, wherein said fungus is afilamentous fungus.
 5. The fungus of claim 4, wherein said filamentousfungus is an Agaricus species.
 6. A fruiting body tissue of the fungusof claim 3, wherein the tissue comprises the expression cassette.
 7. Amethod for expressing a nucleotide sequence in a fungal cell, the methodcomprising: a) transforming a fungal cell with an expression cassette,the expression cassette comprising an isolated regulatory elementoperably linked to a nucleotide sequence wherein the regulatory elementcomprises the nucleotide sequence as set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:4; and b) growing the fungal cell toexpress the nucleotide sequence.
 8. The method of claim 7, wherein theisolated regulatory element initiates expression of the nucleotidesequence and achieves expression in a fruiting body tissue cell.
 9. Themethod of claim 7, further comprising regenerating a stably transformedfungus from the fungal cell; wherein expression of the nucleotidesequence alters a phenotype of the fungus.
 10. The method of claim 9,wherein said fungus is a filamentous fungus.
 11. The method of claim 10,wherein said filamentous fungus is an Agaricus species.
 12. The methodof claim 9, wherein a fruiting body tissue produced by the funguscomprises the expression cassette.